Muscle fibre type composition in young and racing Swedish cold-blooded trotters

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1 Comparative Exercise Physiology 6(1); doi: /s Muscle fibre type composition in young and racing Swedish cold-blooded trotters Kristina Karlström 1, *, Arne Lindholm 2, Eje Collinder 2 and Birgitta Essén-Gustavsson 1 1 Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden 2 Mälaren Equine Clinic, Sigtuna, Sweden * Corresponding author: kristina.karlstrom@kv.slu.se Submitted 25 September 2008: Accepted 6 April 2009 Research Paper Abstract Fibre type composition of skeletal muscle in horses varies due to factors like breed, age and training. Variations due to these factors in muscle fibre type composition of the Swedish cold-blooded trotter, a heavier and slower breed than the Standardbred trotter, have not previously been reported. The objective of this study was therefore to investigate muscle fibre type composition of young and racing cold-blooded trotters using both histochemical and immunohistochemical methods. Muscle biopsies (gluteus medius) were obtained from 2-year-old (n ¼ 18) and 4 8-year-old horses (n ¼ 6). Four of the 2-year-old horses were sampled again during their 4-yearold season. All of the horses were in professional training and the group included superior racehorses. Fibre types (I, IIA and IIB) were visualized with the myosin ATPase technique. Some samples were also stained immunohistochemically to identify MHCI, MHCIIA and MHCIIX fibres using myosin heavy chain (MHC) antibodies. The oxidative capacity of the fibres was subjectively evaluated from NADH-tetrazolium reductase stains. Type I and type IIA fibres were all identified as MHCI and MHCIIA fibres, respectively, whereas type IIB fibre population included both pure MHCIIX and hybrid MHCIIAX fibres. The older racehorses had a higher proportion of type MHCIIA and a lower proportion of type MHCIIX fibres than the 2-year-old horses. Areas of type I fibres were larger and those of type IIB fibres were smaller in racing horses compared with young horses. The proportion of type MHCIIX fibres that stained medium for oxidative capacity increased with age and training. In conclusion, training and racing induce muscular adaptations in cold-blooded trotters as in other breeds. Furthermore, immunohistochemical methods detect hybrid fibres indicating that transitions of fibre types may occur. Keywords: cold-blooded trotters; muscle; oxidative capacity; immunohistochemistry; fibre types Introduction With the biopsy technique, it is possible to study fibre type composition in muscle of horses and differences have been observed between breeds 1. Horses (Quarter horses, Thoroughbreds, Standardbreds) that perform at faster speeds have somewhat more type II fibres than horses that perform at slower speeds (Andalusians, Arabs), although all horses have a high proportion of type II fibres. Many studies report an influence from age and training on muscle fibre composition of horses 2 7. In these studies, fibre type composition was evaluated by staining for myosin ATPase and three fibre types were identified: slow-contracting type I, fast-contracting type IIA and IIB fibres. Type IIA/IIB ratios increased with age and training, and training not growth was shown to be the main factor for this increase. Oxidative capacity was high in type I and IIA fibres, but varied markedly in the type IIB fibres due to the training status. It has also been shown that horses with high performance levels have muscles with higher IIA/IIB ratios and higher oxidative capacities in the type IIB fibres than horses with lower performance levels 3,7 9. Another method of characterizing fibre types is to use myosin heavy chain (MHC) antibodies that identify qcambridge University Press 2009

2 28 the different MHC isoforms MHCI, MHCIIA and MHCIIX. Nowadays this method is often used since a more detailed picture of the muscle and its adaptations to different kinds of stimuli can be obtained With this method, it is possible to detect hybrid fibres, i.e. fibres that contain more than one MHC isoform, which may be an indication that fibres are transforming from one type to another, e.g. from MHCIIX to MHCIIA to MHCI. Using the myosin ATPase method, it is not possible to identify hybrid fibres and small adaptations in the muscle might be overlooked. Studies on both endurance types of horse, Thoroughbreds and Standardbred trotters, have shown that the muscle of trained individuals contains hybrid fibres 12,14,15. One breed of horse, where muscle characteristics have, as yet, not been described is the Swedish cold-blooded trotter. This is a breed that has evolved from farm horses that worked in the forest and the fields. The farmers used to come together to compete with their horses, often on frozen lakes in winter. Today there are farming and trotting types of coldblooded horses, the trotting type being somewhat less heavy than the working horse. The cold-blooded trotter is smaller (height at the withers cm) and more heavily and squarely built (weight c. 500 kg) than the Standardbred trotter. Both these breeds race over short distances, but the speed of the cold-blooded trotter (,1.20 min km 21 or,12.5 m s 21 ) is slower than that of Standardbred trotter (,1.10 min km 21 or,14.5 m s 21 ). The aim was to investigate fibre type composition and oxidative capacity of fibre types in muscles of young and racing cold-blooded trotters that were in professional training, using both histochemical and immunohistochemical techniques. Materials and methods Horses Twenty-four Swedish cold-blooded trotter stallions in professional training were included. Eighteen horses were 2 years old and four of these were sampled again during their 4-year-old season. Six horses were 4 8 years old. The young horses were just at the beginning of their training and the older horses were actively training and racing. Horses usually start racing in the spring of their 3-year-old season. Some of the horses are among the elite in Scandinavia. Muscle biopsies (gluteus medius) were taken at the training camp, in all cases by the same person, using a biopsy needle 16. The samples were obtained at approximately the same relative depth in the muscle, usually 4 5 cm, a similar site as used earlier in our studies on Standardbred trotters. Samples were rolled in talcum powder and frozen in liquid nitrogen and then stored at 2 808C until analysed. This study was approved by the Ethical Committee for Animal Experiments in Stockholm, Sweden. Histochemical analysis Transverse sections, 10 mm, were cut in a cryostat (Reichert-Jung, Cambridge Instruments GmbH, Nussloch, Germany) and stained for myosin ATPase at ph 4.6 to detect fibre types I, IIA and IIB 17. Serial sections, 10 mm, from 2-year olds (n ¼ 7) and 4 8-year olds (n ¼ 10) were also reacted with myosin heavy chain (MHC) antibodies N2-261 (MHC I þ IIA), A4-74 (MHC IIA) and S5-8H2 (MHC I þ IIX) using the PAP staining method. Furthermore, sections were stained with the NADH tetrazolium reductase method 18. To evaluate ATPase fibre type composition and fibre type area, a computerized image analyser (Bio-Rad, Scan Beam, Hadsund, Denmark) was used. The immunohistochemical sections were photographed and fibres were classified according to the MHC content into MHCI, MHCIIA, MHCIIAX and MHCIIX fibres. Oxidative capacity was subjectively evaluated from the NADH stains (30 50 fibres of each type) into high- medium- or low-oxidative fibres. Statistical analyses Differences between groups were evaluated using Mann Whitney U and between staining techniques using Wilcoxon signed-rank tests. Values are presented as median and range. Results K Karlström et al. Fibre types and fibre areas ATPase type I and type IIA fibres corresponded to MHCI and MHCIIA fibres (Table 1). There were fewer pure type MHCIIX (32%, range 25 47) than type IIB fibres (43%, range 29 57; P, 0.001). Some type IIB fibres immunohistochemically contained MHCIIA isoform, and thus hybrid MHCIIAX fibres were identified (Table 2). Within the MHCIIAX fibres, a variation of staining intensity was found (Fig. 1). The oldest racing horses had more type IIA and MHCIIAX fibres and fewer MHCIIX fibres as compared with the youngest (Tables 1 and 2) and a higher MHCIIA:MHCIIX fibre ratio (old: 1.7, range ; young: 1.0, range ; P ¼ 0.02). Three of the four horses (nos 1 3) that were sampled both at 2 and 4 years old had increased the proportion of MHCIIAX fibres at 4 years of age after they had been racing several times (Table 3). The fourth horse lost his 3-year-old season due to injury and did not

3 Muscle fibre types in cold-blooded trotters 29 Table 1 Median (range) for fibre type composition and fibre type areas in skeletal muscle of young horses in training and older cold-blooded trotters actively racing, and significance levels for differences between groups In training 2 years old (n ¼ 18) 4 8 years old (n ¼ 9) Significance level ATPase fibre type (%) I 15 (6 28) 12 (9 27) 0.37 IIA 40 (30 46) 45 (36 54) 0.01 IIB 44 (32 58) 38 (29 48) 0.10 ATPase fibre type area (mm 2 ) I 1961 ( ) 2397 ( ) 0.03 IIA 2447 ( ) 2597 ( ) 0.90 IIB 4193 ( ) 3674 ( ) 0.02 Mean area 3199 ( ) 2874 ( ) 0.11 change in fibre type composition. Areas of the type IIB fibres were smaller and the type I fibres larger in the racing horses as compared with the younger horses (Table 1). Oxidative capacity Oxidative capacity was, in both young and old horses, high in type I and IIA fibres and medium in MHCIIAX fibres. In MHCIIX fibres, the staining intensity for oxidative capacity varied markedly (Fig. 2). In the 2-year-old horses 51% (range 45 54) of MHCIIX had medium staining intensity, and in the 4-year-old horses 81% (range ) were medium stained (Table 4). The remaining MHCIIX fibres had low staining intensity. In the oldest, racing horses all MHCIIX fibres were found to have a medium staining intensity (Fig. 2). race over the same distances as Standardbred trotters ( m). The results suggest that adaptations in the muscle occur due to long-term training as trained and raced horses had a higher oxidative capacity, a higher percentage of type IIA fibres and lower percentage of type IIB fibres, larger type I fibres and smaller type IIB fibres. The immunohistochemical results of the cold-blooded trotters further suggest that the higher percentage of MHCIIA fibres seen with training/ racing is due to a transition from MHCIIX fibres as hybrid MHCIIAX fibres were found. These results are in agreement with other training studies on other breeds that indicate that transitions occur from pure Discussion The results of this study show that fibre type composition of younger and older Swedish cold-blooded trotters is quite similar to that of the Standardbred trotters 12,20,21. This would have been expected since the cold-blooded trotters train in a similar way and Table 2 Median (range) for MHC and m-atpase fibre type composition in skeletal muscle of young horses in training and older cold-blooded trotters actively racing, and significance levels for differences between groups In training 2 years old (n ¼ 7) 7 8 years old (n ¼ 4) Significance level MHC fibre type (%) I 22 (8 28) 10 (9 18) 0.22 IIA 38 (29 46) 50 (43 54) 0.02 IIAX 5 (0 11) 8 (5 11) 0.11 IIX 38 (32 47) 28 (25 32) 0.06 ATPase fibre type (%) I 22 (8 28) 10 (9 18) 0.22 IIA 39 (30 46) 50 (44 54) 0.01 IIB 43 (32 57) 36 (29 44) 0.34 FIG. 1 Photomicrographs of serial sections of a 4-year-old horse. MHCIIAX fibres are marked in all stains. (A) MHCIIA isoform staining; (B) m-atpase ph 4.6; (C) NADH tetrazolium reductase. Bar in (C) (lower right) represents 100 mm

4 30 Table 3 Number of starts and myosin heavy chain fibre type composition of muscle of four horses at 2 and 4 years of age K Karlström et al years 4 years 2 years 4 years 2 years 4 years 2 years 4 years Number of starts MHC fibre type (%) I IIA IIAX IIX type MHCIIX via hybrid MHCIIAX to MHCIIA fibres 10,12,19. The horse material in this study is not large but it is unique in comparison with many other studies, as it included superior racehorses. Some of these horses had been training and racing for several years. This is FIG. 2 Photomicrographs of NADH stains from the same horse at (A) 2 and (B) 4 years old and (C) one 8 year old horse. Bar in (C) (lower right) represents 100 mm in contrast to many earlier training studies where the training has been undertaken for relatively short (5 12 weeks) periods using sedentary horses In these studies, fibre type changes were not seen. In the present study, the youngest horses had a few hybrid fibres and these horses had been in training for a very short period. The 4-year-old horse that had been out of training due to injury also proved to have a low percentage of MHCIIAX fibres (Table 3). A higher amount of hybrid MHCIIAX fibres was seen in the 4-year-old horses that had been in training for a couple of years and had started to race. The staining intensity from MHCIIA isoform varied among MHCIIAX fibres. This may indicate that the fibres are in different states of transformation. The older racehorses that had been training and racing over several years had a high percentage of MHCIIA fibres and only a few hybrid MHCIIAX fibres. This is probably due to the fact that a total transformation of several MHCIIX to pure MHCIIA fibres had occurred by that time. Muscle fibres are recruited in a specific order with type I and IIA fibres first and as the intensity and duration of work increase, more fibres need to be recruited and thus type IIB (MHCIIX) fibres will be included 25,26. The latter are shown to be of great importance to successful racing 27. The size of type IIB fibres was decreased in the older racehorses, which is in agreement with studies on trained and racing Standardbred trotters 3,28. The advantage of a decreased fibre size is that it facilitates transport of oxygen and substrates to and efflux of waste products such as lactate and heat from the fibre. When type IIB (MHCIIX) fibres with a low oxidative capacity get recruited at the end of a race it is likely that they fatigue quickly. All MHCIIX fibres in older horses showed a medium oxidative capacity, which indicates a high degree of fatigue resistance. Transformation of type IIA to type I fibres seemed not to occur in this group of horses since no changes in the percentage of type I fibres were seen. The variation between individuals in type I fibre proportions at 2 years of age was also seen at 4 years of age, which confirms the reliability of muscle biopsy sampling (Table 3).

5 Muscle fibre types in cold-blooded trotters 31 Table 4 Oxidative capacity of the MHC fibre types subjectively classified into high, medium or low according to staining intensity in the NADH tetrazolium reductase stain In training 2 years old (n ¼ 4) 4 years old (n ¼ 4) 7 8 years old (n ¼ 4) Oxidative capacity High Medium Low High Medium Low High Medium Low I þ IIA (%) IIAX (%) IIX (%) Conclusion In Swedish cold-blooded trotters, muscular adaptations occur due to training and racing, and with immunohistochemical methods hybrid fibres were detected, indicating fibre type transitions. Acknowledgements This study was supported by grants from the Swedish Board (ATG), Hälsinge Trav AB and Mälaren Equine Clinic. The authors thank trainer Jan-Olov Persson for excellent help in performing this study. References 1 Snow DH and Guy PS (1981). Fiber type and enzyme activities of the gluteus medius in different breeds of horses. In: Poortmans J and Niset G (eds) Biochemistry of Exersice IV-B. 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